Saliva is vital for oral health. It is essential for the hydration of the oral mucosa; it provides lubrication, begins nutrient digestion and imparts antimicrobial and mechanical protection for the mouth and upper gastrointestinal tract. Reduced flow of saliva (xerostomia) greatly impact the quality of life of suffers. A key event in the secretagogue stimulation of both the fluid and protein component of saliva is an increase in the cytosolic free calcium concentration [Ca2+]i. This increase in [Ca2+]i has specific temporal and spatial characteristics which are thought to be important for appropriately activating effectors important for the secretion of saliva. Acetylcholine and the majority of other secretagogues act in a metabotropic manner stimulating the production of inositol 1,4,5-trisphosphate (InsPS) and the release of intracellular Ca2+. In salivary acinar cells, Ca2+ signals can also be generated by an alternative, largely independent ionotropic process following adenosine triphosphate (ATP) activation of the P2X class of Ca2+ permeable ion channels. The latter mechanism would be predicted to result in Ca2+ signals and effector activation with distinct spatial and temporal characteristics. Using contemporary optical techniques such as digital imaging, total internal reflection microscopy and confocal microscopy combined with transgenic animal models and specific pharmacology we will expand on findings generated in isolated salivary gland acini to define Ca2+ signaling events stimulated through both metabotropic and in particular through ionotropic P2X4 purinergic receptor (P2X4R) stimulation in an organotypic parotid gland slice preparation (aim 1). Ca2+ signals as a result of P2X4R activation are profoundly enhanced following protein kinase A (PKA) activation. Since fluid secretion is also enhanced when PKA is activated this may be the physiologically relevant situation. In specific aim 2 we propose to define the molecular mechanism underlying this event. In specific aim 3 a primary goal will be to assess whether ionotropic purinergic signaling through P2X4 receptors (P2X4R), either alone, or following specific pharmacological manipulation is an effective activator of the fluid and exocytotic secretion mechanism. These studies are designed to advance our understanding of the complex series of regulated molecular events which result in fluid secretion and may ultimately lead to the design of therapeutic strategies for the treatment of xerostomia based on exploiting the physiology of remaining functional acinar tissue.